Introduction:Escherichia coli is one of many species of bacteria living in the lower intestines of mammals, known as gut flora. When located in the large intestine, it assists with waste processing, vitamin K production, and food absorption. Discovered in 1885 by Theodor Escherich, a German pediatrician and bacteriologist, E. coli are abundant: the number of individual E. coli bacteria in the faeces that a human defecates in one day averages between 100 billion and 10 trillion. However, the bacteria are not confined to the environment, and specimens have also been located, for example, on the edge of hot springs. The E. coli strain O157:H7 is one of hundreds of strains of the bacterium that causes illness in humans. E. coli are unable to sporulate. Thus, treatments which kill all active bacteria, such as pasteurization or simple boiling, are effective for their eradication, without requiring the more rigorous sterilization which also deactivates spores. As a result of their adaptation to mammalian intestines, E. coli grow best in vivo or at the higher temperatures characteristic of such an environment, rather than the cooler temperatures found in soil and other environments. The enteric E. coli (EC) are divided on the basis of virulence properties into enterotoxigenic (ETEC – causative agent of diarrhea in humans, pigs, sheep, goats, cattle, dogs, and horses), enteropathogenic (EPEC – causative agent of diarrhea in humans, rabbits, dogs, cats and horses); enteroinvasive (EIEC – found only in humans), verotoxigenic (VTEC – found in pigs, cattle, dogs and cats); enterohaemorrhagic (EHEC – found in humans, cattle, and goats, attacking porcine strains that colonize the gut in a manner similar to human EPEC strains) and enteroaggregative E. coli (EAggEC – found only in humans). E. coli O157:H7 was first recognized as a pathogen as a result of an outbreak of unusual gastrointestinal illness in 1982. The outbreak was traced to contaminated hamburgers, and the illness was similar to other incidents in the United States and Japan. The etiologic agent of the illness was identified as a rare O157:H7 serotype of Escherichia coli in 1983. This serotype had only been isolated once before, from a sick patient in 1975.
Intended Use:genesig Kit for Escherichia coli O157:H7 (E.coli_O157) genomes is designed for the in vitro quantification of E.coli_O157 genomes. The kit is designed to have a broad detection profile. Specifically, the primers represent 100% homology with over 95% of the NCBI database reference sequences available at the time of design. The dynamics of genetic variation means that new sequence information may become available after the initial design. Primerdesign periodically reviews the detection profiles of our kits and when required releases new versions.This primers used to develop this kit have been adapted from the recent paper below
Li et al. BMC Microbiology (2017) 17:215; Multiplex real-time PCR assay for detection of Escherichia coli O157:H7 and screening for non-O157 Shiga toxin-producing E. coli.
Dynamic Range of Test:Under optimal PCR conditions genesig E.coli_O157 detection kits have very high priming efficiencies of >90% and can detect less than 100 copies of target template
Suitable Sample Material:All kinds of sample material suited for PCR amplification can be used. Please ensure the samples are suitable in terms of purity, concentration, and RNA/DNA integrity. Always run at least one negative control with the samples. To prepare a negative control, replace the template DNA sample with RNase/DNase free water.
Test Principle:Real-time PCR A E.coli_O157 specific primer and probe mix is provided and this can be detected through the FAM channel. The primer and probe mix provided exploits the so-called TaqMan® principle. During PCR amplification, forward and reverse primers hybridize to the E.coli_O157 DNA. A fluorogenic probe is included in the same reaction mixture which consists of a DNA probe labeled with a 5`-dye and a 3`-quencher. During PCR amplification, the probe is cleaved and the reporter dye and quencher are separated. The resulting increase in fluorescence can be detected on arange of qPCR platforms.
Kit Components:
E.coli_O157 specific primer/probe mix (150 reactions)FAM labelled
E.coli_O157 positive control template
Internal extraction control primer/probe mix (150 reactions)VIC labelled as standard
Internal extraction control DNA (150 reactions)
Endogenous control primer/probe mix (150 reactions)FAM labelled
RNase/DNase free water, for resuspension of primer/probe mixes
Template preparation buffer, for resuspension of internal control template, positive control template and standard curve preparation
Reagents and Equipment to be Supplied by the User:Real-Time PCR InstrumentDNA extraction kitThis kit is recommended for use with genesig Easy DNA/RNA extraction kit
However, it is designed to work well with all processes that yield high quality DNA with minimal PCR inhibitors
Pipettors and TipsVortex and CentrifugeThin walled 1.5ml tubesqPCR plates
Storage and Stability:This kit is stable at room temperature but should be stored at -20ºC on arrival. Once the lyophilized components have been resuspended they should not be exposed to temperatures above -20ºC for longer than 30 minutes at a time and unnecessary repeated freeze/thawing should be avoided. The kit is stable for six months from the date of resuspension under these circumstances.
